Collagen is the most common protein in the structural support of the human or mammalian body. Collagen's basic elemental unit is the tropocollagen protein. Tropocollagen is composed of three polypeptide chains of the same size. These chains are wound about each other forming a superhelical cable or a triple-stranded helical rod. Each of the three chains in tropocollagen consists of about a thousand amino acid residues.
Five different types of collagen proteins are currently recognized as distinct, differing in amino acid composition and length. Type I collagen is composed of two alpha-1(I) and one alpha-2 polypeptide chains. Type I collagen is mostly found in the supporting structure of skin tissue, tendon, bone and in the eye cornea. Type II collagen contains three polypeptide chains of the alpha-1(II) type and is found primarily in articular cartilage, within the intervertebral discs and in the vitreous body within the eye. Type III collagen is composed of three alpha-1(III) polypeptide chains and is found in tissues such as fetal skin, the cardiovascular system and reticular fibers in the eye. Type IV collagen has a mixture of two alpha-1(IV) and one alpha-2(IV) polypeptide chains and is primarily found in basement membranes. Finally, Type V collagen has two alpha-1 (V) and one alpha-2(V) polypeptide chains and is found, e.g. in placenta and skin. A comparison of the human alpha-1(II), bovine alpha-1(II) and bovine alpha-1(I) amino acid sequences is provided in Appendix A. The bovine sequences are partial.
Rheumatoid arthritis is a cell-mediated autoimmune disease, i.e. a condition where the immune system mistakenly perceives the body's own tissue as foreign and mounts an abnormal immune response against it. Rheumatoid arthritis is characterized by persistent inflammatory synovitis that causes destruction of cartilage and bone erosion, leading to structural deformities in the peripheral joints. Joints containing articular cartilage of which Type II collagen is a major component are particularly affected.
Rheumatoid arthritis is accompanied by joint swelling, inflammation, stiffness and pain especially upon flexing. In the advanced stages of arthritis, debilitating pain may result from even a slight movement of the joints. A substantial percentage of afflicted humans possess T-cells of the CD4+ type specifically reactive with collagen and/or have an abnormal humoral response against collagen.
Present treatment for arthritis involves use of nonspecific cytotoxic immunosuppressive drugs. These drugs suppress the entire immune system and are incapable of selectively suppressing the abnormal autoimmune response. This global restraint of the immune system over time increases the risk of infection. Non-limiting examples of such immunosuppressive drugs include methotrexate, cyclophosphamide, Imuran (azathioprine) and cyclosporin A.
Additionally, prolonged therapy with these nonspecific cytotoxic immunosuppressive drugs entails toxic side effects, including increased tendency towards development of certain malignancies, kidney failure, diabetes and liver function disorders. Moreover, cytotoxic immunosuppressive drug therapy merely slows down the progress of the disease, which resumes at an accelerated pace after the therapy is discontinued. For example, about six weeks after such a drug is discontinued, the patient deteriorates to the same stage as before the treatment was begun. In addition, effectiveness of these drugs is self-limiting; they gradually cease being effective after about 2-5 years.
Steroid compounds such as prednisone and methylprednisolone (which are also non-specific immunosuppressive and anti-inflammatory drugs) are also used for symptomatic relief. Steroids also have significant toxic side effects associated with their long term use.
Thus, current treatments for arthritis are of limited efficacy, involve significant toxic side effects, and cannot be used indefinitely. Rheumatoid arthritis afflicts over 2 million individuals in the United States in any given year. Accordingly, there is an acute need for novel treatments and for novel therapeutic compositions for human autoimmune arthritis that do not suffer from one or more of the drawbacks identified above.
An alternative treatment for arthritis is the oral antigen tolerization therapy proposed by the present inventors. It involves the oral, enteral, or by-inhalation administration of one or more tissue-specific antigens (i.e. antigens occurring only in the tissue under autoimmune attack) which have the ability to suppress the autoimmune response responsible for a particular autoimmune disease specifically, thus leaving other immune functions essentially intact.
The antigens useful in this approach generally include autoantigens, i.e. tissue-specific antigens that are themselves the subject of autoimmune attack. Bystander antigens, which are also tissue-specific (but are not the target of autoimmune attack) also possess the ability to elicit suppressor T-cells which are targeted to the afflicted tissue where they exert their immune suppressive activity via the release of transforming growth factor-beta (TGF-.beta.). TGF-.beta. in turn down-regulates all immune cells concentrated in the vicinity of the afflicted tissue, thereby suppressing immune responses in that locality. Bystander antigens include without limitation portions of autoantigens that (i) are not themselves the target of autoimmune attack and (ii) possess the requisite suppressive activity via elicitation of suppressor T-cells.
Prior to the work of the present inventors, oral antigen tolerance therapy for arthritis had been tried only on artificially induced arthritis-like diseases in animals (adjuvant arthritis and collagen-induced arthritis).
Regardless of the correlation that may exist between human autoimmune arthritis and its animal (rodent) models, oral, enteral or inhalatory antigen tolerization had never been tried in humans afflicted with arthritis. Moreover, in the rodent models, collagen was shown only to prevent disease induction and had no or minimal effect on pre-induced disease.
The role of collagen in arthritis and its models, has been the subject of many publications, including some describing the oral use of collagen to prevent disease induction in the rodent model. A chronological summary of the state of the art follows.
Collagen was shown to be the tissue under autoimmune attack in the artificially induced cell-mediated autoimmune arthritis model by Trentham, D. E. et. al., J. Clin. Invest., 66:1109-1117, November, 1980. The authors showed that both humoral and cellular autoimmunity to type I and type II collagen is a feature of both adjuvant- and collagen-induced arthritis in rats.
Schoen, R. T. et. al., J. Immunol. 128:717-719, February, 1982, found that type I collagen, unlike type II collagen, coupled artificially to naive spleen cells and injected into normal animals was ineffectual in preventing inducement of arthritis-like disease.
Thompson, H. S. G. et. al., Clin. Exp. Immunol., 64:581-586, 1986, reported resistance to induction of a polyarthritis model in rats after prophylactic intragastric administration of soluble type II collagen protein (2.5 or 25 .mu.g/g body weight/day for 5 days). The lower amount, 2.5 .mu.g/g, was slightly more effective.
Nagler-Anderson, C. et. al., Proc,. Natl. Acad. Sci. USA, 83:, October, 1986, pp. 260-262, that prophylactic intragastric administration (500 .mu.g/mouse/12 times in 6 weeks) of undenatured soluble type II collagen was found to suppress inducement of arthritis in DBA/1Lac J mice by challenge with an adjuvant and collagen mixture.
Myers, L. K. et. al., J. Immunol., 143:3976-3980, December, 1989, that the intravenous administration of type II but not type I collagen protein would confer resistance to subsequently induced arthritis-like disease in animal models. In a subsequent publication, the same group, J. Exp. Med., 170:1999-2010, December, 1989, cite three other references for the same proposition: Schoen et. al., Supra; Cremer et. al., J, Immunol., 87:2995, 1983; and Englert et. al., Cell. Immunol,, 87:357, 1984.
In this same J. Exp. Med. publication the authors state that they have identified a fragment of chicken type II collagen (CB11 corresponding to amino acid residues 122-147) which upon intravenous administration to mice prior to challenge conferred protection against collagen induced arthritis. This peptide had the sequence P-T-G-P-L-G-P-K-G-Q-T-G-E-L-G-I-A-G-F-K-G-E-Q-G-P-K [SEQ ID NO: 1].
In summary, the most relevant art teachings are limited as follows:
experiments were conducted solely on induced animal models; PA1 animals tested were limited to species susceptible to disease induction; PA1 the treatment was ineffective against already induced disease; PA1 intravenous administration of type I collagen was ineffective to prevent disease inducement. PA1 one immunosuppressive epitope of chick type II collagen intravenously administered to mice was identified.